HSTA Inquiry schedule: Clinical Chemistry with Dr. ShiemkeDaily Schedule: Students' Weeks:
Monday morning: Discussion of goals of the enquiry and introduction of the techniques used in the laboratory.
Monday Afternoon: Creatine kinase assay, data analysis and identification of samples with abnormal CK activity.
Tuesday Afternoon: Creatine kinase electrophoresis on cellulose acetate.
Wednesday (all day): Total serum cholesterol assay, data analysis and identification of abnormal samples. Lipoprotein electrophoresis on cellulose acetate
Thursday Afternoon: Repeat any experiments with uncertain results. Fun with chemistry (turn copper penny to gold, rip aluminum can in half with bare hands, colored flames, etc.)
Goal
In this laboratory experience we will learn about the structure and function of proteins through their use in diagnosis of diseases in the clinical laboratory. We will use techniques similar to those used in most clinical labs to explore how creatine kinase is used to diagnose heart attacks and how cholesterol levels in blood are measured. We will also measure the levels of HDL (good cholesterol) and LDL (bad cholesterol) in samples.
Proteins are large complex molecules found in all living organisms. They act primarily as catalysts, speeding up reactions that are required for the organism to live. In fact proteins are responsible for catalyzing every reaction that takes place in a living organism and are referred to as the work-horse of life. Proteins that catalyze reactions are referred to as enzymes. Many other proteins do not catalyze reactions but act as carriers of small molecules, antibodies in host defense and provide structure to cells and organs.
Proteins are polymers, which means they are long linear molecules composed of many smaller molecules (monomers) linked end-to-end. The small molecule monomers used to make proteins are called amino acids. There are 20 different kinds of amino acids used to make proteins. These amino acids differ from each other in size, shape, charge, and chemical properties. Proteins can contain anywhere from ~100 to several thousand amino acids monomers. The different chemical nature of the monomers gives these proteins an enormous diversity in structure and function, depending on which amino acids are used in the protein and in which order they are put together.
Inside the living organism the proteins don’t exist as long straight chain molecules. After the amino acids are linked together the proteins fold into a compact structure that is nearly spherical. For each protein the final structure will be different, because each protein has its unique sequence of amino acids that compose it. This is illustrated in Figure 1.
Figure 1........................................................................ Figure 2
In figure 2 the sequence of amino acids for a small protein (ribonuclease A) is shown. This protein has 124 amino acid monomers linked together to form the final polymer. In the figure, each amino acid is represented by a circle, and the letter inside the circle is a code that identifies each amino acid. For example amino acid 1 is K (lysine), 2 is E (glutamic acid), 3 is T (threonine) etc.
One of the features that makes cells from different organs different from each other is the different types of proteins found in each type of cell. For example, some of the proteins found in a liver cell will be different from those found in a heart cell. This is because some of the chemistry done in liver cells is different from that done in heart cells, and different proteins are neede to carry out these different reactions. In disease states cells of a certain organ will die and release their contents to the blood. So if heart cells die, in a heart attack for example, there will be proteins in the blood released from the heart cells that are not normally found there. Identification of these proteins and measuring their amount can often provide crucial clues in diagnosis of disease. We will perform these type of measurements in this laboratory experience, examining the levels of creatine kinase and cholesterol in samples and making conclusions from these measurements about whether these samples are normal or indicative of possible disease states.
Creatine Kinase
Creatine kinase (CK) is an enzyme found principally in skeletal muscle, heart, and brain. CK catalyzes the following reaction:
The creatine phosphate in this reaction is used as a form of stored energy. When a burst of energy is needed in the cell the creatine phosphate is used to convert ADP to ATP and the ATP is used as a source of energy for many types of reactions.
As mentioned above, CK is found primarily in muscle and heart cells. A small amount is always present in the blood, but after an injury to heart or skeletal muscle the amount of CK in the blood can increase approximately 10-fold within 18-36 hours of the injury. We will use a method called an enzyme assay to measure CK levels in samples and compare them to a normal standard to determine which (if any) have elevated CK levels.
An enzyme assay takes advantage of the catalytic properties of the enzyme. That is, if a small amount of an enzyme can convert a certain amount of a substrate to a product in a given time, then a larger amount of enzyme can produce a proportionally larger amount of product over the same time period. In the case of CK we could measure the amount of either creatine or ATP produced from creatine phosphate and ADP. However, both creatine and ATP are difficult to measure directly, so we will use a coupled assay, in which one of the products of the CK reaction is a substrate for subsequent reactions, which produce an easily measured product. In this particular case the coupled reactions are shown below.
In the final reaction, glucose-6-phosphate dehydrogenase (G-6-PD) catalyzes the conversion of glucose-6-phosphate and NAD to 6-phosphogluconate and NADH. NADH is very easy to measure since it strongly absorbs light of 340 nm. So we will combine our samples with a mixture that contains all the components shown in the scheme above, except CK. The CK present in the sample will initiate the sequence of reactions culminating in production of NADH and we will use a spectrometer to measure the NADH, which will be proportional to the amount of CK present in the original sample.
Samples containing abnormally high levels of CK will be further analyzed by electrophoresis. Electrophoresis is a technique in which a mixture of proteins is placed in a porous medium (a gel) and the gel is placed in a strong electric field. Proteins are charged molecules so they will migrate toward to oppositely charged electrode. The rate of migration of a protein through the gel will depend on its size and charge. Large proteins experience more friction as they move so they migrate slower. Proteins with more charges will experience a greater attraction to the oppositly charged electrode so they will move faster. This is illustrated in Figure 3.
Figure 3
The CK protein found in skeletal muscle is slightly different in amino acid sequence from the CK found in Heart. These slightly different forms of an enzyme are called isozymes. As a result of their differences in structure the CK from skeletal muscle (CK3) can be separated from the CK in heart (CK2) by electrophoresis. So by applying the samples containing high CK levels to electrophoresis we can determine if the elevated level is due to abnormally high levels of CK2 or CK3. Samples from heart attack victims have abnormally high levels of CK2. This method is one of the common methods used in clinical labs to diagnose heart attacks.
Cholesterol is a type of fat molecule found in most cell membranes. Cholesterol must be transported through the blood stream from the places it is synthesized to the locations where it is used. In the blood stream , cholesterol is transported by proteins called lipoproteins. Cholesterol is found primarily in two types of lipoproteins called high-density lipoprotein (HDL) and low-density lipoprotein (LDL). HDL contains more protein and less cholesterol than LDL. HDL is thought to contribute less to hardening of the arteries (atherosclerosis) than LDL, so HDL is often referred to as good cholesterol and LDL is bad cholesterol. In this part of the laboratory experience we will again use a coupled enzyme assay to measure amounts of cholesterol in samples. We will then examine those samples with high cholesterol levels by electrophoresis to separate HDL and LDL and determine if the high cholesterol is due to elevated HDL or LDL.
The coupled assay for cholesterol is illustrated below.
In the scheme above, CE is cholesterol esterase. Cholesterol is bound to the lipoproteins in the form of ester molecules with fatty acids. CE catalyzes the disruption of the ester bond between the cholesterol molecule and the fatty acid, giving free cholesterol. CO is cholesterol oxidase, which catalyzes the oxidation of cholesterol by oxygen, generating hydrogen peroxide (H 2O 2). The peroxidase enzyme catalyzes the conversion of aminoantipyrene to a dye that strongly absorbs light of 505 nm so it can be easily detected in a spectrometer. We will add our samples to a mixture that contains all of the above components, except cholesterol. As with the CK assay, the amount of the final product produced (the dye) will be proportional to the amount of cholesterol present in the sample.
HDL and LDL differ in amino acid sequence, so they can be separated by electrophoresis. Samples found to have high cholesterol levels will be examined by electrophoresis to determine whether the high cholesterol is due to elevated HDL (good for health) or elevated LDL (bad for health).
Through these laboratory exercises the student will obtain an understanding of what proteins are, what they do and how they are used in the clinical laboratory to diagnose disease. The lab techniques are relatively easy to master and are in fact similar to those used in clinical labs in the recent past. Now the analyses are all automated, but the principles used by the automated instruments are the same as those used in these laboratory exercises.
If time permits we will also explore some entertaining laboratory procedures. For example, I will demonstrate how to tear an aluminum can in half with my bare hands. I will also show the students how to turn a copper penny to gold, and how to make a spooky green flame.
Developed on July 11, 2005 by Sohail Khan
